Localization of Decorin in Leptin –Treated Traumatic Oral Ulcer in Rats

Sabrin S. Abd, B.D.S., H.D.D., MSC Department of Oral Diagnosis, College of Dentistry, University of Baghdad/Iraq.

Nada M. Al-Ghaban, B.D.S., M.Sc., Ph.D. Department of Oral Diagnosis, College of Dentistry, University of Baghdad/Iraq.

Abstract Back ground: The oral mucosa mainly exposed to injury by trauma or pathologic diseases, Leptin is a hormone known to has many physiological roles that effects the cell function and, acts as wound healing accelerator. The aim of the present study is to evaluate the effect of topical application of recombinant leptin on induced traumatic oral ulcer healing by mean of immunohistochemical localization of Decorin. Materials and methods: Forty eight male Albino rats age between 2-3 months with body weight between (200.45-270.53g),were subjected for traumatic ulcer by surgical blade on the right side of the buccal mucosa by surgical blade (no.15) ,with diameter of (8 mm). The animals divided into two groups ;control group : the ulcer treated with sterilized distal water, the experimental group: the ulcer treated with 10µl of 1 µg/ml recommbenant leptin.The rats were sacrificed at 3,7,10 days. Immunohistochemical methods were used to detect the expression of decorin in both control and study groups . Results: The present study showed that the recompenant leptin treatment increased expression of decorin in ulcer area from the 3rd day of ulceration by epithelial cell, endothelial cells, fbroblast cells , with highly significant differences in comparison with control group . Conclusion : leptin accelerated the healing process in oral mucosa ulcer by increase the expression of decorin in early healing period than control.

Key words: leptin, oral mucosa, ulcer, Decorin

INTRODUCTION Any wound healing is a series of biological processes, include migration, adhesion, proliferation, and differentiation of several cell types. All these activities are triggered by chemo-attraction of the cells; polypeptide mediators bind to their cell-surface receptors , integrins bind to extracellular matrix components, and different growth factors regulate different cell functions. The process ending with the formation and maturation of a new extracellular matrix (1,2,3) . The extracellular matrix (ECM) contains a collection of molecules that regulate both structural integrity and function of the cell (4). The most extensively studied member of the ECM class is decorin which is a small leucine-rich proteoglycan (SLRP) (5) . Decorin interact with a variety of different ligands including; other ECM constituents, cellular receptors, growth factors, proteases, and other signaling molecules; to regulates different cellular processes (6,7) including angiogenesis (8,9) innate immunity (10), inflammation (11,12) , fibrosis(13) ,wound healing (14) . Decorin has ability to activate or inhibit receptor signaling (15), and isolates the growth factors (16). Leptin, is a 16 kDa anti-obesity hormone produced predominantly by adipose tissues and secreted into the blood stream as a free , or as a protein. In addition to its influences on the body weight homeostasis (17). Its also exhibit a different physiological actions such as hematopoiesis (18), bone formation (19), angiogenesis (20), and wound healing (21). The multi-functionality of leptin and it plays a variety of physiological roles not only as a systemic hormone but also as a local growth factor (22).

MATERIALS AND METHODS Forty eight albino rats weighting (200-270 gm), aged (2-3) months were used in this study. They maintained under control conditions of temperature, drinking and food consumption .All experimental procedures were carried out in accordance with the animal experimentation ethical principles of the Biotechnical Research Center at Al-Nahrain University. Induction of oral ulcer Ulceration of the oral mucosa of each rats in this study were done by the following steps; First the animal anesthetize via intrapretoneal injuction of ketamine (50 mg/kg) and xylazine (5 mg/kg) (23). The mucosal ulceration with 8 mm diameter was performed on the right side of the buccal mucosa by abarasion

with a surgical scalpel blade (no.15) (24). The control group(24 rats): the ulcers treated with 10µl of sterilized distal water. While the Experimental group(24 rats):the ulcers treated with 10µL of 1µg/ml recombinant leptin protein from Abcam company UK (ab646). Then the animals were sacrificed according to 3 healing intervals into 3,7, and 10 days (16 rats from both groups in each periods). Then the specimens from each rats were taken and prepared for histological (H&Estain) and for immunohistochemical localization of decorin by using of Anti- Decorin antibody, Rabbit polyclonal, (ab175404) , and detection kit (ab80436) from Abcam company UK. Determination of immunohistochemical results for Decorin Under light microscope at x40, a five fields were chosen from epithelium area, and another five fields were chosen from connective tissue from each tissue section, captured by digital camera and the images evaluated imported to computer. The evalution of staining results was achieved by applying Aperio positive pixel count algorithms program (from Aperio Image Scope software v11.1.2.760 (Aperio Technologies Inc, USA)),we neglected the weak positive reading in yellow color . The average of mean positive percentages for each five area were obtained and considered as the value of expression of Decorin per slide

RESULT: Decorin expression At 3rd day in control group, weak positive reactivity to decorin was seen in suprabasal layer of the new front epithelium and stromal cells of lamina properia(Fig.1A&B) .In the study group, strong positive membranous expression for decorin were seen in spinosum layer of the new epithelium and in collagen fibers and stromal cells of lamina properia (Fig.1C&D) . At 7th day in the control group, moderat positive expression for decorin was detected in the spinosum and granulosum layers of new epithelium as well as in fibroblast cells, endothelial cells, and in collagen fibers of lamina properia (Fig.2A&B). In the study group ,strong positive reaction to the decorin can be observed in spinosum and granulosum layers of epithelium and in endothelial cells, fibroblasts and collagen fibers of lamina properia (Fig2 C&D). At 10th day in the control group, strong reactivity to the decorin was obeviously seen in the epithelium.The lamina properia also showed strong expression in the collagen fibers and endothelial

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cells(Fig. 3A&B). In the study group, weak positive expression was seen in the granulosum layer of epithelium tissue only. In the lamina properia was limited to the collagen fibers and fibroblast (Fig.3 C&D). Statistical analysis of immunohistochemical result The mean difference between control and study group was illusetrated in Table-1, which showed highly significant differences between study and control group in the expression of decorin in epithelial cells at 3rd day and 10th day , and significant at 7th day. For the lamina properia ,the result showed significant differences between study and control group in the expression of decorin in all healing periods . Table-2 showed that there were highly significant difference between epithelium and lamina proeria in expression of decorin in control group at 3rd and 10th day , and significant at 7th day . For study group the result revealed highly significant differences between epithelium and lamina properia in expression of decorin at 3rd day and significant differences between them in 7th and 10th days . Regarding to the duration differences in each control and study group, the ANOVA test was used as shown in Table-3. For control group both the epithelium and lamina properia showed highly significant differences.For study group, epithelium showed highly significant differences between duration ,while in lamina properia showed non - significant difference.

Figure1 A: Decorin expression at 3rd day in epithelium of control group

x40.

Figure1B: Decorin expression at 3rd day in lamina prperia of control

group x40.

Figure 1 C : Decorin expression at 3rd day in study group in epithelium

tissue x40

Figure1D: Decorin expression at 3rd day in study group in lamina properia

x40.

Figure 2.A: Decorin expression at 7th day in control group in epithelium

tissue x40.

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Figure 2B: Decorin expression at 7th day in control group in lamina

prperia x40.

Figure 2.C: Decorin expression at 7th day in study group in epithelium

tissue x40.

Figure 2.D: Decorin expression at 7th day in study group in lamina

properia X40.

Figure3.A: Decorin expression at 10th day in control group in epithelium

tissue X40

Figure3.B: Decorin expression at 10th day in control group in lamina

properia.

Figure3.C: Decorin expression at 10th day in study group in epithelium

tissue.

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Figure3.D: Decorin expression at 10th day in study group in lamina

prperia.

Table 1: Groups' comparison for Positive cells expressed decorin in both epithelium and lamina properia at each duration

P-value

T-test

Study Control Site Day

SD Mean SD Mean 0.000 HS 7.184 10.35 35.65 4.519 8.375 Epithelium 3rd 0.012 S 3.343 10.92 28.67 4.709 11.68 Lamina properia

0.004 S 4.133 9.368 36.98 4.230 23.51 Epithelium 7th 0.010 S 3.505 7.059 34.015 7.230 19.981 Lamina properia

0.001 HS 5.343 9.552 15.256 9.304 36.731 Epithelium 10th 0.094 S 1.939 5.776 36.51 7.992 29.771 Lamina properia

Table 2:Sites’ comparisons for Positive cells expressed decorin in both

groups at each duration P-value T-test SE Mean different Group Day 0.001 HS 5.836 3.443 -20.09 Control 3rd 0.000 HS 16.195 1.350 21.863 Study

0.013 S 3.304 3.763 -12.427 Control 7th 0.030 S 2.719 2.773 7.543 Study

0.001 HS 5.588 4.290 23.975 Control 10th 0.094 S 1.909 0.999 1.907 Study

Table 3: ANOVA test for duration differences in epithelium and

lamina properia in both groups P-value F-test Groups Marker

P

REFERENCES 1. CILLO C, CANTILE M, FAIELLA A, BONCINELLI E. 2001. Homeobox

genes in normal and malignant cells. J Cell Physiol.188:161-169. 2. CONWAY EM, COLLEN D, CARMELIET P. 2001. Molecular mechanisms

of blood vessel growth. Cardiovasc Res. 49:507-521. 3. ALPISTE-ILLUECA FM, BUITRAGO-VERA P, DE GRADO-

CABANILLES P, FUENMAYOR-FERNANDEZ V, GIL-LOSCOS FJ. 2006. Periodontal regeneration in clinical practice. Med Oral Patol Oral CirBucal.11:E382-92.

4. IOZZO R.V. 1997. The family of the small leucine-rich proteoglycans: keyregulators of matrix assembly and cellular growth, Crit. Rev. Biochem. Mol.Biol. 32:141–174.

5. IOZZO RV; SCHAEFER L. 2015. "Proteoglycan form and function: Acomprehensive nomenclature of proteoglycans". Matrix Biology. 42: 11–55. 11.

6. BRYSON JM, PHUYAL JL, SWANV, CATERSON AD. 1999. Leptin hasacute effects on glucose and lipid metabolism in both lean and goldthioglucose-obese mice. Am J Physiol., 277: E417–E422.

7. ICHII M., FRANK M.B., IOZZO R.V., KINCADE P.W. 2012. The canonical Wnt pathway shapes niches supportive of hematopoietic stem/progenitor cells, Blood. 119:1683–1692.

8. NIKOLOVSKA K., RENKE J.K., JUNGMANN O., GROBE K., IOZZOR.V., ZAMFIR A.D. 2014. A decorin-deficient matrix affects skinchondroitin/dermatan sulfate levels and keratinocyte function, MatrixBiol.35:91–102.

9. JÄRVELÄINEN H., SAINIO A., WIGHT T.N.2015. Pivotal role for decorinin angiogenesis, Matrix Biol.43:15–26.

10. NEILL T., PAINTER H., BURASCHI S., OWENS R.T., LISANTI M.P.,SCHAEFER L., ET AL. 2012. Decorin antagonizes the angiogenic network.Concurrent inhibition of Met, hypoxia inducible factor-1α and vascular endothelial growth factor and induction of thrombospondin-1 and TIMP3, J.Biol. Chem. 287: 5492–5506.

11. FREY T., SCHROEDER N., MANON-JENSEN T., IOZZO R.V.,SCHAEFER L. 2013. Biological interplay between proteoglycans and theirinnate immune receptors in inflammation, FEBS J. 280: 2165–2179.

12. BOCIAN C., URBANOWITZ A.K., OWENS R.T., IOZZO R.V., GOTTE M.,SEIDLER D.G. 2013. Decorin potentiates interferon-gamma activity in amodel of allergic inflammation, J. Biol. Chem. 288:12699–12711.

13. BORGES M.C., NARAYANAN V., IOZZO R.V., LUDWIG M.S. 2015.Deficiency of decorin induces expression of Foxp3 in CD4(+) CD25(+) T cells in a murine model of allergic asthma, Respirology. 20: 904–911.

14. BAGHY K., IOZZO R.V., KOVALSZKY I. 2012. Decorin-TGFβ axis in hepatic fibrosis and cirrhosis, J. Histochem. Cytochem. 60:262–268.

15. Järveläinen H., Puolakkainen P., Pakkanen S., Brown E.L., Höök M., Iozzo R.V., et al. 2006. A role for decorin in cutaneous wound healing andangiogenesis, Wound Repair Regen. 14:443–452.

16. DELLETT M., HU W., PAPADAKI V., OHNUMA S. 2012. Small leucinerich proteoglycan family regulates multiple signalling pathways in neuraldevelopment and maintenance, Develop. Growth Differ. 54 : 327–340.

17. K. BAGHY, P. TATRAI, E. REGOS, I. KOVALSZKY. 2016. Proteoglycans in liver cancer, World J. Gastroenterol. 22: 379–393.

18. BRYSON JM, PHUYAL JL, SWANV, CATERSON AD. 1999. Leptin hasacute effects on glucose and lipid metabolism in both lean and goldthioglucose-obese mice. Am J Physiol. 277: E417–E422.

19. GAINSFORD T, WILLSON TA, METCALF D, HANDMAN E,MCFARLANE C, ET AL. 1996. Leptin can induce proliferation,differentiation, and functional activation of hemopoietic cells. Proc NatlAcadSci U S A. 93: 14564–14568.

20. KUME K, SATOMURA K, NISHISHOU S, KITAOKA E, YAMANOUCHIK, ET AL. 2002. Potential role of leptin in endochondral ossification. JHistochemCytochem.,50:159–169.

21. Bouloumie´ A, Drexler HC, Lafontan M, Busse R Leptin, the product of Obgene, promotes angiogenesis. Circ Res. 1998, 83: 1059–1066.

22. MURAD A, NATH AK, CHA ST, DEMIR E, FLORES-RIVEROS J, ET AL.2003. Leptin is an autocrine/paracrine regulator of wound healing. FASEB J., 17: 1895–1897.

23. KERIMOĞLU G, YULUĞ E., KERIMOĞLU S., ÇITLAK A. 2013. Effects of leptin on fracture healing in rat tibia. Eklem Hastalık Cerrahisi Joint Diseases and Related Surgery. 24 (2):102-107.

24. GALYLÉIA MENESES CAVALCANTE, RENATA JANAÍNA SOUSA DEPAULA, LEONARDO PERES DE SOUZAI, FABRÍCIO BITU SOUSA,MÁRIO ROGÉRIO ,LIMA MOTA, ANA PAULA NEGREIROS NUNESALVES. 2011. Experimental Model Of Traumatic Ulcer In The Cheek Mucosa Of Rats. Acta Cirúrgica Brasileira.Vol. 26 (3).

25. KINSELLA MG, FISCHER JW, MASON DP, WIGHT TN. 2000. Retrovirally mediated expression of decorin by macrovascular endothelialcells. Effects on cellular migration and fibronectin fibrillogenesis in vitro. JBiol Chem. 275: 13924–32.

26. KRESSE H, SCHONHERR E. 2001. Proteoglycans of the extracellular matrix and growth control. J Cell Physiol. 189: 266–74.

27. OKSALA O, SALO T, TAMMI R, HÄKKINEN L, JALKANEN M, INKI P,LARJAVA H J. 1995. Histochem Cytochem. Expression of proteoglycans and hyaluronan during wound healing. 43(2):125-35.

28. ALAIN SIMÉON, YANUSZ WEGROWSKI, YANNICK BONTEMPS,FRANÇOIS-XAVIER MAQUART. 2000. Expression of Glycosaminoglycans and Small Proteoglycans in Wounds: Modulation by the Tripeptide–Copper Complex Glycyl-L-Histidyl-L-Lysine-Cu2+. Journal of InvestigativeDermatology. Volume 115, Issue 6, 962-968.

29. FLEISCHMAJER R, FISHER LW, MACDONALD ED, JACOBS L,PERLISH JS, AND TERMINE JD.1991. Decorin interacts with fibrillarcollagen of embryonic and adult human skin. J Struct Biol. 106: 82.

30. Katerina Nikolovska, Jana K. Renke, Oliver Jungmann, Kay Grobe, Renato V. Iozzo, Alina D. Zamfir, Daniela G. Seidler . 2014. A decorin-deficient matrixaffects skin chondroitin/dermatan sulfate levels and keratinocyte function. Elsevier B.V., Matrix Biology. 35 : 91–102.

31. JORDI XAUS, MO`NICA COMALADA, MARINA CARDO´ , ANNABELF. VALLEDOR, ANTONIO CELADA. 2001. Decorin inhibits macrophagecolony-stimulating factor proliferation of macrophages and enhances cellsurvival through induction of p27Kip1 and p21Waf1. The American Society of Hematology. volume 98;7.

32. ZHANG G1, EZURA Y, CHERVONEVA I, ROBINSON PS, BEASON DP,CARINE ET, SOSLOWSKY LJ, IOZZO RV, BIRK DE. 2006. Decorin regulates assembly of collagen fibrils and acquisition of biomechanicalproperties during tendon development. J Cell Biochem. 15;986:1436-49.

33. KALAMAJSKI S, OLDBERG A. 2010. The role of small leucine-rich proteoglycans in collagen fibrillogenesis. Matrix Biol. 29:248–53.

34. LASSI NELIMARKKA, HELI SALMINEN, TEIJO KUOPIO, SEPPONIKKARI, TAUNO EKFORS, JUKKA LAINE, LAURI PELLINIEMI,HANNU JA¨RVELA¨INEN. 2001. Decorin Is Produced by CapillaryEndothelial Cells in Inflammation-Associated Angiogenesis. American Journal of Pathology.vol. 158, No. 2.

35. SCHONHERR E, SUNDERKOTTER C, IOZZO RV, SCHAEFER L. 2005. Decorin, a novel player in the insulin-like growth factor system. J Biol Chem;280: 15767–72.

36. MANORANJAN SANTRA, SUTAPA SANTRA, JING ZHANG ANDMICHAEL CHOPP, Ectopic decorin expression up-regulates VEGFexpression in mouse cerebral endothelial cells via activation of thetranscription factors Sp1, HIF1a, and Stat3. J. Neurochem. 2008, 105, 324–337.

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